PROJECT SUMMARY/ABSTRACT Scientific Premise: The platelet release reaction is central to thrombosis, hemostasis, and thus is critical for vascular function. Although this reaction permits platelets to control micro-environments at damaged vascular sites, we have scarce knowledge of the structural elements that regulate the extrusion of platelet cargo from ?- granules and allow its incorporation into a growing thrombus. Thus, ultrastructural studies of resting and agonist-activated mouse and human platelets are needed to gain new insights into the infrastructure of this process. Specific Aim 1: Map ?-granule release within the hemostatic thrombus to determine the relative distributions of granule-PM, granule-granule, and granule-canalicular system (CS) fusion events. We hypothesize that the events underlying granule fusion differ between the core vs. the shell of a growing thrombus. We will generate high-resolution, 3D models of platelets at progressive stages of activation within the growing thrombus to precisely define membrane fusion events and thus the mechanism of cargo release in distinct regions of the thrombus. Specific Aim 2: Determine how spatial the distribution/association of ?-granule cargo proteins regulate the kinetics of release. We reported that von Willebrand factor (VWF) and fibrinogen (Fg) map to non-overlapping zones within the same ?-granule (Pokrovskaya et al., 2017a), demonstrating the potential for cargo packaging heterogeneity. In this Aim, we will define the distribution of other ?-granule cargo within the granule matrix using immunogold labeling/super-resolution microscopy and biochemistry to map ?protein neighborhoods?. The findings will shed light on how packaging structures regulate cargo release rates. Specific Aim 3: Determine, in vivo, how neck/pipe dynamics affect agonist-induced release rates. These experiments will utilize live-cell imaging techniques to define the dynamics of pipes/necks and explicitly demonstrate their regulation of agonist-induced cargo release for the first time. Impact: A research priority of the American Society of Hematology is to understand clot formation. Despite this focus, we lack clear pictures of how platelet cargo release is controlled by spatial, temporal, and agonist signals and the contributions of these signals to thrombosis at the site of vascular damage. We need an ultrastructural view of the platelet cargo release process as the framework to understand these physiological events. Otherwise, the design of therapies to control platelet activity and hemostasis lack a rational and predictive conceptual framework. To meet this challenge, our project concentrates on ?-granules, the largest and most abundant storage granule in the platelet and the source of myriad effects on the vascular microenvironment. Our findings will yield new insights into how platelet secretion is spatially and temporally regulated in the growing thrombus.